The invention relates to methods for the expression and secretion of factor VIII polypeptides.
Hemophilia A affects one in every 10,000 males and is caused by a deficiency of the factor VIII protein in the plasma. Based on the level of factor VIII activity in the blood, hemophilia A is categorized into mild, moderate, and severe forms. Fifty percent of hemophilia A patients have the severe form of the disease that is characterized by spontaneous and prolonged bleeding episodes.
Factor VIII is a cofactor in the coagulation pathway. Circulating in the blood, factor VIII is non-covalently complexed with its carrier protein von Willebrand factor. This interaction stabilizes factor VIII and prevents the association of factor VIII with membrane surfaces. The conversion of factor VIII into its active state, factor VIIIa, occurs via the proteolysis of factor VIII by thrombin or factor Xa. The proteolysed factor VIIIa dissociates from von Willebrand factor. A membrane bound complex containing factor VIIIa and factor IXa is formed that subsequently activates factor X in the coagulation cascade.
Currently, hemophilia A is treated by the frequent infusion of purified factor VIII into the blood. While this method of treating hemophilia A does reduce the frequency and severity of bleeding, this therapy is limited by the availability and the cost of purified factor VIII, the short half life of factor VIII in vivo, and the necessity of removing contaminating AIDS and hepatitis viruses. While, recombinant factor VIII is now available, this form of factor VIII maintenance therapy is both expensive and chronic.
Gene therapy is an attractive alternative to the protein infusion treatments for hemophilia A. Two gene therapy approaches may be used. In vivo gene therapy introduces nucleotides encoding the factor VIII protein into the patient""s cells. Ex vivo gene therapy techniques introduce the nucleotides encoding the factor VIII protein into in vitro cultured cells. The transformed cultured cells are subsequently reimplanted into the patient.
Studies of factor VIII biogenesis and secretion have been limited by the lack of human cell lines that express significant amounts of factor VIII. Analysis of secretion has been limited to autologous gene expression. In general, these studies show factor VIII has low expression levels due to unstable RNA and poor secretion. See, for example, Lenting et al. (1998) Blood 92:3983-3996, Connelly et al. (1996) Human Gene Therapy 7:183-195, Kaufman et al. (1989) Mol. Cell. Biol. 9: 1233, Dorner et al. (1987) J. Cell Biol. 105:2665 and the references cited therein. These studies suggest that expression of factor VIII and secretion of factor VIII may be more efficient in cells where it is endogenously expressed.
Factor VIII mRNA is expressed in many tissues including the kidney, spleen, liver, and lymph nodes. Human and canine studies have shown that factor VIII levels rise to normal following liver transplantation, during which there can be no extrahepatic synthesis of factor VIII. This indicates that the liver synthesizes a clinically significant amount of factor VIII protein. It is well known in the art that hepatocytes express factor VIII, however, whether other types of liver cells synthesize factor VIII remains controversial. See, for reviews, Bloom et al. (1979) Clin. Haematol. 8:53-77 and Lenting (1998) Blood 92:3983-3996, both of which are herein incorporated by reference.
Many different gene therapy approaches to treat hemophilia A are currently being studied. Ex vivo gene therapy techniques have found that factor VIII protein expression is low in transformed in vitro cultured cells and undetectable in vivo (Lynch et al. (1993) Hum. Gene Therapy 4:259; Chuah et al. (1995) Hum. Gene Ther. 6:1363; Hoeben et al. (1990) J. Biol. Chem. 265:7318; Hoeben et al. (1993) Hum. Gene Ther. 4:179; Israel et al. (1990) Blood 75:1074 and van der Eb (1996) J. Clin. Biochem. Nutr. 21: 78-80; all of which are herein incorporated by reference).
Both the canine and murine systems are being used to develop in vivo therapy treatment for hemophilia A. Many of these have directed the expression of factor VIII to the liver hepatocytes (Connelly et al. (1998) Blood 91:3273-3281, Connelly et al. (1996) Human Gene Therapy 7:183-195, Connelly et al. (1996) Blood 88:3846, Giles et al. (1982) Blood 60:727). Phenotypic correction of murine hemophilia A was sustained for over 9 months through the in vivo transformation of hepatocytes. However, sustained expression was not uniformly achieved. Instead, factor VIII levels rapidly declined in certain animals over time. Yet, the level of factor VIII mRNA declined only minimally in these animals. Further characterization showed that neither the humoral immune response nor the cell-mediated immune response was responsible for the decline of factor VIII levels in the plasma. Therefore, the level of mRNA in the transformed hepatocytes did not parallel the concentration of factor VIII protein in the plasma (Connelly et al. (1998) Blood 91:3273-3281; all of which are herein incorporated by reference).
The present invention provides an improved method of factor VIII expression by targeting expression of factor VIII to cells that express a key regulatory protein involved in factor VIII biogenesis.
Compositions and methods for the expression of a nucleotide sequence encoding a factor VIII polypeptide or a functional variant thereof are provided. Specifically, the methods of the invention provide for the stable introduction of a nucleotide sequence encoding factor VIII or a functional variant thereof into an isolated LSEC. The genetically modified LSEC is subsequently cultured under conditions that allow for the expression and secretion of the factor VIII polypeptide. In one embodiment of the present invention, the culturing of the genetically modified LSECs occurs in vitro, while in other embodiments, the culturing of the genetically modified LSECs occurs in vivo.
The present invention further provides methods to increase the level of a factor VIII polypeptide or functional variant thereof in the blood stream of a subject. The method comprises the stable introduction of a nucleic acid sequence encoding the factor VIII polypeptide or a functional variant thereof into an isolated LSEC. The genetically modified LSEC is implanted into a subject such that an increased level of factor VIII polypeptide in the blood of the subject occurs. In certain embodiments, the subject is characterized by having factor VIII deficient plasma. In other embodiments, the subject has hemophilia A.
The present invention further provides method to increase the level of a factor VIII polypeptide or a functional variant thereof in the blood stream of a subject through the stable introduction of a nucleic acid sequence encoding the factor VIII polypeptide or a functional variant thereof into an LSEC cultured in vivo. In this method the factor VIII polypeptide is preferentially produced in the LSECs, such that the levels of the factor VIII polypeptide is increased in the blood stream of the subject. In certain embodiments the subject is characterized by having factor VIII deficient plasma. In other embodiments the subject has hemophilia A.
Compositions are also provided for an isolated LSEC having stably incorporated a DNA construct comprising a nucleotide sequence encoding a factor VIII polypeptide or a function variant thereof, operably linked to a promoter active in the LSEC.